拟南芥JAL30基因的克隆及功能研究

发布时间：2018-04-15 14:55

本文选题：凝集素 + 油菜素内酯　；参考：《福建农林大学》2017年硕士论文

【摘要】：凝集素(lectin)是一类从动物和植物中提取的糖蛋白或结合糖的蛋白,能够可逆的、特异的结合糖类、糖蛋白和糖脂表面糖基而不改变糖类的结构。在植物体中,糖蛋白起到信号传递或转换作用,而凝集素可特异识别其糖链残基,两者相互作用,引发下游生化信号级联反应。木菠萝凝集素(Jacalin)是一种植物中特有的具有对多种糖结合特异性的凝集素蛋白,其中拟南芥中的JAL30被报道参与植物对草食动物的化学防御功能。本实验室的前期研究表明,JAL30蛋白在外源油菜素内酯(BrassinosteroidsBRs)处理时含量增加且蛋白翻译后修饰发生变化,然而JAL30应答BR信号通路的分子机制仍不清楚。本文主要对拟南芥中JAL30基因的表达以及功能进行了研究。首先从拟南芥中克隆了JAL3 基因并且构建了 35S启动子驱动的JAL30的cDNA融合GFP的过表达载体,获得了多个转基因株系并对其进行表型观察。结果表明,转基因植株并没有明显的表型变化。我们还购买了JAL30的T-DNA突变体植株,也没有发现植株有明显表型。可能是由于凝集素蛋白主要通过一种短暂应答响应的方式调节植物生长,JAL30过量表达或者缺失对植物的长期生长表型无明显影响。同时我们还将此载体转入烟草进行亚细胞定位研究,瞬时转化实验显示JAL30主要在细胞核以及细胞质中大量表达。研究表明JAL30蛋白对BR以及其他激素诱导剂有应答响应,因此我们对转基因株系进行了 BR以及茉莉酮酸甲酯(JAMe)处理,并从转录和翻译水平进行了表达分析。结果表明,在转录水平上,BR处理并没有明显增加JAL30的表达。在蛋白水平上,BR处理对JAL30有一定的上调作用,但外源施加BR抑制剂PPZ,再施加外源BR处理以后,上调作用就不再明显。表明BR促进了 JAL30的蛋白表达,而BR抑制剂PPZ可能抑制了 JAL30蛋白表达。而JAMe处理则明显上调JAL30蛋白表达。JAMe与植物的防御机制相关,而BR则对植物生长起着重要的作用,后续实验结果表明JAL30对两者都有响应,在植物体内有着重要的功能。本文中对过表达植株进行了免疫共沉淀以及质谱分析(IP-MS)。提取过表达植株蛋白并进行蛋白免疫沉淀实验,再经质谱分析其相互作用蛋白。结果表明,JAL30本身存在三个磷酸化修饰位点,并鉴定到53个相互作用蛋白。实验结果证明JAL30参与到植物体内多种信号通路,自身受到磷酸化翻译后修饰调节。我们也鉴定到多个潜在的相互作用蛋白,为进一步研究JAL30蛋白的功能及调节机制奠定了基础。
[Abstract]:Lectin is a class of glycoproteins or sugar binding proteins extracted from animals and plants, which can be reversible, specific binding carbohydrate, glycoprotein and glycolipid surface glycosyl without changing the structure of carbohydrates.In plant, glycoprotein plays the role of signal transduction or conversion, while lectin can specifically recognize its sugar chain residues, and the interaction between them leads to downstream biochemical signal cascade reaction.Pineapple lectin (Jacalin) is a unique lectin protein that is specific to many kinds of sugar binding in plants. JAL30 in Arabidopsis thaliana has been reported to be involved in plant chemical defense against herbivores.Previous studies in our laboratory showed that the content of JAL30 protein increased during BrassinosteroidsBRs treatment with exogenous BrassinosteroidsBRs, but the molecular mechanism of JAL30 response to Br signaling pathway was still unclear.In this paper, the expression and function of JAL30 gene in Arabidopsis thaliana were studied.Firstly, the JAL3 gene was cloned from Arabidopsis thaliana and the cDNA fusion vector of 35s promoter driven JAL30 was constructed. Several transgenic lines were obtained and their phenotypes were observed.The results showed that there was no obvious phenotypic change in transgenic plants.We also purchased JAL30 T-DNA mutant plants and found no obvious phenotype.It may be that lectin protein regulates the overexpression or absence of JAL30 mainly through a transient response to the growth of plants, and has no significant effect on the long-term growth phenotype of plants.At the same time, we also transferred the vector into tobacco for subcellular localization. Transient transformation experiments showed that JAL30 was mainly expressed in the nucleus and cytoplasm.The results showed that the JAL30 protein was responsive to Br and other hormone inducers, so we treated the transgenic lines with Br and jasmonate methyl ester, and analyzed their expression and expression at the transcriptional and translation levels.The results showed that Br treatment did not significantly increase the expression of JAL30 at the transcriptional level.At the protein level, Br treatment had a certain up-regulation effect on JAL30, but the upregulation was no longer obvious after exogenous Br treatment and exogenous Br treatment.The results showed that Br promoted the expression of JAL30 protein, while Br inhibitor PPZ inhibited the expression of JAL30 protein.JAMe treatment significantly upregulated the expression of JAL30 protein. JAMe was related to the defense mechanism of plants, while Br played an important role in plant growth. The results of follow-up experiments showed that JAL30 was responsive to both of them and had important functions in plants.In this paper, the over-expressed plants were analyzed by immunoprecipitation and mass spectrometry.The expressed plant protein was extracted and the protein immunoprecipitation assay was carried out, and then the interacting protein was analyzed by mass spectrometry.The results showed that there were three phosphorylation sites in JAL30 and 53 interacting proteins were identified.The results showed that JAL30 was involved in many signaling pathways in plants and was regulated by phosphorylation posttranslational modification.We also identified a number of potential interaction proteins, which laid a foundation for further study of the function and regulatory mechanism of JAL30 protein.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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